Composition for preventing or treating cervical cancer having human papillomavirus plasmodium and immunity enhancer

ABSTRACT

A composition for preventing or treating cervical cancer comprising a human papillomavirus plasmodium and an immunity enhancer is provided. A fusion protein including a fusion polypeptide recombined to transform a 3D structure of E6 and E7, which are antigens against types 16 and 18 human papillomavirus (HPV), a signal peptide for secreting the fusion polypeptide outside the cells and an immunity enhancer peptide present in an individual is also provided. The fusion protein may be useful in treating HPV-triggered tumors by inducing an immune response specific to the antigens against the HPV types 16 and 18.

RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national phase application ofPCT/KR2010/005367 (WO 2012/020871), filed on Aug. 13, 2010, entitled“Composition for Preventing or Treating Cervical Cancer Having HumanPapillomavirus Plasmodium and Immunity Enhancer”, which is incorporatedherein by reference in its entirety.

BACKGROUND

1. Field of the Invention

The present invention relates to a composition for preventing ortreating cervical cancer comprising a human papillomavirus (HPV)plasmodium and an immunity enhancer, and, more particularly, to a fusionprotein including a fusion polypeptide recombined to transform a3-dimensional (3D) structure of E6 and E7, which are antigens of types16 and 18 HPV, a signal peptide for secreting the fusion polypeptideoutside the cells, and an immunity enhancer peptide present in anindividual, wherein the fusion protein may treat HPV-triggered tumors byinducing an immune response specific to HPV type 16 and 18 antigens.

Incorporated by reference herein in its entirety is the Sequence Listingentitled “PCT_KR2010_(—)005367_Sequence Listing_ST25-2,” created Apr. 4,2014, size of 11 kilobytes.

2. Discussion of Related Art

Cervical cancer has been known as a disease that develops from infectionof human papillomaviruses (HPVs) of very high concern such as types 16and 18 (zur Hausen, H et al. Biochem Biophys Acta 1996, 1288; F55-F78,Mark H et al. J Natl Cancer Inst 1993, 85; 958-964). Among HPV proteins,E6 and E7 proteins play an important role in the onset of cervicalcancer, and are important target substances used to prepare a vaccinefor treating and preventing cervical cancer since they are confirmed tobe expressed in 99% of tumor tissues from cervical cancer patients (vonKnebel Doeberitz et al. Int. J. Cancer 1992, 51; 831-834). In this case,E6 is bound to p53 known as a tumor suppressor protein to facilitatedegradation of the p53, thereby obstructing a cell cycle from leading tothe apoptosis pathway, and E7 is bound to a retinoblastoma protein, pRb,known as a tumor suppressor factor to inactivate the retinoblastomaprotein and facilitate degradation of the retinoblastoma protein,thereby allowing the cell cycle to enter the S stage (Cobrinik et al.,Trends Biochem Sci 1992, 17:312-5).

In clinical trials using a composition including a nucleic acid sequencein which HPV16 E6 and E7 proteins are simultaneously expressed to treatcervical cancer, however, the composition has shown a poor therapeuticeffect (Garcia F et al. Obstet Gynecol 2004, 103; 317-326). Theseresults indicate that a sufficient antigen-specific immune response totreat or suppress cervical cancer is not triggered when only an HPVantigen is simply administered.

Therefore, it is necessary to enhance the immunogenicity of E6 and E7proteins to treat cervical cancer, and remove the proteins' capabilityof developing into cancer.

SUMMARY OF THE INVENTION

The present invention is directed to providing a novel fusion proteinfor preventing or treating HPV-triggered diseases, and a polynucleotideencoding the fusion protein. Here, the fusion protein suppresses the HPVE6 and E7 proteins' capability of developing into cancer and showsenhanced immunogenicity as well.

Also, the present invention is directed to providing a recombinantvector expressing the fusion protein, a host cell including therecombinant vector, and a method of expressing the fusion protein usingthe host cell.

In addition, the present invention is directed to providing acomposition for preventing or treating HPV-triggered diseases using thefusion protein.

Furthermore, the present invention is directed to providing a method ofpreventing or treating HPV-triggered diseases using the composition.

One aspect of the present invention provides a fusion protein includinga fusion polypeptide configured to transform a 3D structure of E6 and E7derived from HPV types 16 and 18 and having an amino acid sequence setforth in SEQ ID NO: 1, a signal peptide for secreting the fusionpolypeptide, and an immunity enhancer peptide.

Another aspect of the present invention provides a polynucleotideencoding the fusion protein according to one exemplary embodiment of thepresent invention.

Still another aspect of the present invention provides a recombinantvector including the polynucleotide according to one exemplaryembodiment of the present invention.

Still another aspect of the present invention provides a host celltransformed with the recombinant vector according to one exemplaryembodiment of the present invention.

Still another aspect of the present invention provides a method ofexpressing the fusion protein of the present invention by incubating thehost cell transformed with the recombinant vector according to oneexemplary embodiment of the present invention.

Still another aspect of the present invention provides a composition forpreventing or treating an HPV-triggered disease in an individual in needthereof. Here, the composition includes at least one selected from thegroup consisting of the fusion protein according to one exemplaryembodiment of the present invention, the host cell transformed with therecombinant vector that expresses the fusion protein, and a homogenateof the host cell as an effective ingredient.

Yet another aspect of the present invention provides a method ofpreventing or treating HPV-triggered diseases in an individual in needthereof. Here, the method includes administering an effective amount ofthe composition according to one exemplary embodiment of the presentinvention to the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentinvention will become more apparent to those of ordinary skill in theart by describing in detail exemplary embodiments thereof with referenceto the attached drawings, in which:

FIG. 1 is a graph showing the HPV16 E6-specific CD8+ T cellularresponses which are induced by treatment with GX-188E according to oneexemplary embodiment of the present invention after a tumor cell line,TC-1, is injected subcutaneously into C57BL/6 rats in a model ofanticancer treatment.

FIG. 2 is a graph showing the HPV16 E7-specific CD8+ T cellularresponses which are induced by treatment with GX-188E according to oneexemplary embodiment of the present invention after a tumor cell line,TC-1, is injected subcutaneously into C57BL/6 rats in the model ofanticancer treatment.

FIG. 3 is a graph showing the HPV18 E6-specific CD8+ T cellularresponses which are induced by treatment with GX-188E according to oneexemplary embodiment of the present invention after a tumor cell line,TC-1, is injected subcutaneously into C57BL/6 rats in the model ofanticancer treatment.

FIG. 4 is a graph showing the HPV18 E7-specific CD8+ T cellular responsewhich is induced by treatment with GX-188E according to one exemplaryembodiment of the present invention after a tumor cell line, TC-1, isinjected subcutaneously into C57BL/6 rats in the model of anticancertreatment.

FIG. 5 is a graph showing the anti-cancer effects which are caused bytreatment with GX-188E according to one exemplary embodiment of thepresent invention after a tumor cell line, TC-1, is injectedsubcutaneously into C57BL/6 rats in the model of anticancer treatment.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, exemplary embodiments of the present invention will bedescribed in detail. However, the present invention is not limited tothe embodiments disclosed below, but can be implemented in variousforms. The following embodiments are described in order to enable thoseof ordinary skill in the art to embody and practice the presentinvention.

Although the terms first, second, etc. may be used to describe variouselements, these elements are not limited by these terms. These terms areonly used to distinguish one element from another. For example, a firstelement could be termed a second element, and, similarly, a secondelement could be termed a first element, without departing from thescope of exemplary embodiments. The term “and/or” includes any and allcombinations of one or more of the associated listed items.

It will be understood that when an element is referred to as being“connected” or “coupled” to another element, it can be directlyconnected or coupled to the other element or intervening elements may bepresent. In contrast, when an element is referred to as being “directlyconnected” or “directly coupled” to another element, there are nointervening elements present.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of exemplaryembodiments. The singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises,”“comprising,” “includes” and/or “including,” when used herein, specifythe presence of stated features, integers, steps, operations, elements,components and/or groups thereof, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components and/or groups thereof.

With reference to the appended drawings, exemplary embodiments of thepresent invention will be described in detail below. To aid inunderstanding the present invention, like numbers refer to like elementsthroughout the description of the figures, and the description of thesame elements will be not reiterated.

Hereinafter, configurations of the present invention will be describedin further detail.

The present invention is directed to providing a novel fusion proteinincluding a fusion polypeptide configured to transform a 3D structure ofE6 and E7 derived from HPV types 16 and 18 and having an amino acidsequence set forth in SEQ ID NO: 1, a signal peptide for secreting thefusion polypeptide, and an immunity enhancer peptide.

The fusion protein according to one exemplary embodiment of the presentinvention may include a fusion polypeptide recombined to transform a 3Dstructure of the E6 and E7 derived from the HPV types 16 and 18. Moreparticularly, the fusion polypeptide is a fusion polypeptide in which1^(st) to 85^(th) amino acids of the E6 protein derived from the HPVtype 16, 1^(st) to 65^(th) amino acids of the E7 protein, 71^(st) to158^(th) amino acids of the E6 protein, and 51^(st) to 98^(th) aminoacids of the E7 protein, 1^(st) to 85^(th) amino acids of the E6 proteinderived from the HPV type 18, 1^(st) to 65^(th) amino acids of the E7protein, 71^(st) to 158^(th) amino acids of the E6 protein, and 51^(st)to 105^(th) amino acids of the E7 protein are bound in sequence.

Most particularly, the fusion polypeptide may have an amino acidsequence set forth in SEQ ID NO: 1.

Also, the signal peptide refers to a peptide including approximately 20to 30 amino acids, which serves to secrete a protein expressed in cells,particularly, a protein including a fusion polypeptide of E6 and E7outside the cells. Also, a nucleic acid sequence encoding the signalpeptide is referred to as a “secretory signal sequence.” The fusionpolypeptide of E6 and E7 according to one exemplary embodiment of thepresent invention is a protein (i.e., a nucleus protein) expressed inthe nuclei of the cells infected with viruses, and thus shows weakimmunity. Therefore, the signal peptide expressed from the secretorysignal sequence induces secretion of E6 and E7 antigens whose 3Dstructure is transformed outside the cells, which leads to an increasein antigen-specific humoral and cellular immune responses.

A signal peptide used in higher eukaryotic cells may be used as thesignal peptide. For example, a secretory signal sequence of tissueplasminogen activator (tPa), HSV gDs or a growth hormone may be used.More preferably, a tPa may be used. Most preferably, the signal peptidehas an amino acid sequence set forth in SEQ ID NO: 2.

Also, the immunity enhancer peptide refers to a peptide that serves toenhance an immune response by activating cells associated with theimmune response (for example, dendritic cells, etc.).

A CD40 ligand, an fms-like tyrosine kinase-3 (Flt3) ligand, flagellin,or OX40 may be used as the immunity enhancer peptide. More preferably,the Flt3 ligand may be used herein. The Flt3 ligand is a factor forinducing proliferation and maturation of dendritic cells (DCs), whichmay enhance an immune response against an antigen and show an excellenteffect to relieve a tumor when fused with a tumor antigen. Mostpreferably, the Flt3 ligand may have an amino acid sequence set forth inSEQ ID NO: 3.

Also, the present invention is directed to providing a polynucleotideencoding the fusion protein according to one exemplary embodiment of thepresent invention.

The polynucleotide encodes the fusion protein according to one exemplaryembodiment of the present invention. Here, the fusion polypeptide of E6and E7 may be encoded from a base sequence set forth in SEQ ID NO: 4,but the present invention is not limited thereto. Also, the signalpeptide may be encoded from a base sequence set forth in SEQ ID NO: 5,but the present invention is not limited thereto. The immunity enhancerpeptide may be encoded from a base sequence set forth in SEQ ID NO: 6,but the present invention is not limited thereto.

Also, the polynucleotide according to one exemplary embodiment of thepresent invention may be prepared using chemical synthesis or a geneticengineering technique. The chemical synthesis is known in the relatedart, and any method may be used herein. Also, the polynucleotide may besynthesized using commercially available nucleic acid synthesis and maybe purchased from a nucleic acid supplier. When the polynucleotide isprepared using the genetic engineering technique, the polynucleotide maybe prepared, for example, by separately constructing nucleic acidfragments encoding a fusion polypeptide of E6 and E7, a signal peptideand an immunity enhancer peptide as known in the prior art and bindingthese fragments according to a frame thereof. A method of preparing thenucleic acid fragment is widely known in the related art. Therefore, aperson having ordinary skill in the art may easily bind the nucleic acidfragments using a proper restriction enzyme. According to a specificembodiment of the present invention, a method of preparing apolynucleotide using the chemical synthesis is disclosed.

In addition, the present invention is directed to providing arecombinant vector including the polynucleotide according to oneexemplary embodiment of the present invention.

In the present invention, the term “vector” refers to a gene constructincluding an exogenous DNA fragment which is inserted into the genome toencode a polypeptide. A vector associated with the present invention isa vector in which a secretory signal sequence, a nucleic acid sequenceencoding a fusion polypeptide of E6 and E7 whose HPV 3D structure istransformed, and a nucleic acid sequence encoding the immunity enhancerpeptide are inserted into the genome. Examples of the vector may includea plasmid vector, a cosmid vector, a bacteriophage vector, a yeastvector, or a viral vector such as an adenoviral vector, a retroviralvector or an adeno-associated viral vector.

The secretory signal sequence is a nucleic acid sequence encoding apeptide that can secrete a tumor antigen expressed in the cells outsidethe cells to recognize immunocytes. For example, the secretory signalsequence may include a secretory signal sequence of tPa, HSV gDs, or agrowth hormone. Preferably, a secretory signal sequence used in highereukaryotic cells including a mammal, and, more preferably, the tPa maybe used herein. Most preferably, the secretory signal sequence may havea base sequence set forth in SEQ ID NO: 5. Also, the secretory signalsequence according to one exemplary embodiment of the present inventionmay be substituted with a codon having a high expression frequency, andused in a host cell.

Also, the nucleic acid sequence encoding the immunity enhancer peptiderefers to a nucleic acid sequence encoding a peptide that enhances animmune response by activating cells associated with the immune response(for example, dendritic cells, etc.). A CD40 ligand, an Flt3 ligand,flagellin, or OX40 may be used as the immunity enhancer peptide. Morepreferably, the Flt3 ligand may be used as the immunity enhancerpeptide. Also, the nucleic acid sequence encoding the immunity enhancerpeptide according to one exemplary embodiment of the present inventionmay be substituted with a codon having a high expression frequency, andused in a host cell.

In addition, the polynucleotide included in the recombinant vectoraccording to one exemplary embodiment of the present invention may besubstituted with a codon having a high expression frequency in the hostcell. Among codons commanding amino acids when DNA is transcribed andtranslated into a protein in a host cell, there are codons having highpreference, depending on the host. As used in the present invention, theterm “being substituted with a codon having a high expression frequencyin a host cell” or “codon-optimized” refers to a state in which apolynucleotide is substituted with theses codons having high preferenceto increase the expression efficiency of amino acids or a proteinencoded by nucleic acids of the polynucleotide.

Here, the “host cell” includes a prokaryotic or eukaryotic cell. In thiscase, the eukaryotic cell includes a lower eukaryotic cell includingthose of fungi or yeast, as well as a higher eukaryotic cell includingthose of mammals.

The recombinant vector according to one exemplary embodiment of thepresent invention may include a nucleic acid sequence encoding thefusion protein in a suitable form to express the nucleic acid sequenceencoding the fusion protein of the present invention in a host cell.That is, the recombinant vector according to one exemplary embodiment ofthe present invention includes one or more regulatory sequences selectedbased on a host cell that may be used for expression. Here, theregulatory sequences may be operably coupled to a nucleic acid sequenceto be expressed.

The term “being operably coupled” refers to a state in which a desirednucleotide sequence (for example, in an in vitrotranscription/translation system or a host cell) is coupled to theregulatory sequence in a suitable manner to express the desirednucleotide sequence.

The term “regulatory sequence” refers to a sequence including apromoter, an enhancer and another regulatory element (for example, apolyadenylation signal). The regulatory sequence includes a sequencecommanding constitutive expression of a desired nucleic acid sequence inmany host cells, and a sequence commanding expression of a desirednucleic acid sequence only in a certain host cell (for example, atissue-specific regulatory sequence). A person having ordinary skill inthe art may understand that the design of an expression vector may varyaccording to factors such as selection of a host cell to be transformed,and a level of expression of a desired protein. The expression vectoraccording to one exemplary embodiment of the present invention may beintroduced into a host cell to express the fusion protein.

Also, the expression vector according to one exemplary embodiment of thepresent invention may be, for example, prepared using the standardrecombinant DNA technology. For example, the standard recombinant DNAtechnology includes ligation of blunt and cohesive termini, treatmentwith a restriction enzyme to provide a proper terminus, removal of aphosphate group through treatment with alkaline phosphatase to preventinappropriate binding, and enzymatic binding using T4 DNA ligase. Theexpression vector according to one exemplary embodiment of the presentinvention may be prepared by recombining DNA encoding a signal peptideobtained by chemical synthesis or genetic recombination technology, DNAencoding a fusion polypeptide of HPV E6 and E7, and DNA encoding animmunity enhancer peptide with a vector including a proper regulatorysequence. The vector including the regulatory sequence may be purchasedor prepared in a commercially available fashion. In the presentinvention, a vector for preparing a DNA vaccine, that is, pGX27, wasprepared for use.

According to one exemplary embodiment, the recombinant vector of thepresent invention may be used to prepare a cell line for producing thefusion protein according to one exemplary embodiment of the presentinvention, or may be used as a vector for transferring a gene in genetherapy, or a pharmaceutically active ingredient which itself isadministered to an individual.

Also, the present invention is directed to providing a host celltransformed with the recombinant vector according to one exemplaryembodiment of the present invention.

The kind of the host cell is as listed above.

The transformation may be performed using a known method of introducinga nucleic acid sequence into an organism, a cell, a tissue or an organ.In this case, a method that may be used may be selected so that it canbe suitable for a host cell within the scope of the present invention tobe understood at a level of a person having ordinary skill in the art.For example, such a method includes electroporation, protoplast fusion,calcium phosphate (CaPO₄) precipitation, calcium chloride (CaCl₂)precipitation, agitation using a silicon carbide fiber,agrobacteria-mediated transformation, PEG, dextran sulfate, andlifofectamin, but the present invention is not limited thereto.

Also, the present invention is directed to providing a method ofexpressing a fusion protein according to one exemplary embodiment of thepresent invention by incubating the transformed host cell according toone exemplary embodiment of the present invention.

The fusion protein according to one exemplary embodiment of the presentinvention may be easily expressed and mass-produced by incubating thetransformed host cell in a proper medium, or introducing the transformedhost cell into any animal and incubating the transformed host cell invivo.

In addition, the present invention is directed to providing acomposition for preventing or treating an HPV-triggered disease in anindividual in need thereof. Here, the composition includes at least oneselected from the group consisting of the fusion protein according toone exemplary embodiment of the present invention, a host celltransformed with a recombinant vector expressing the fusion protein, anda homogenate of the fusion protein as an effective ingredient.

In the present invention, the term “individual” includes a mammal suchas a human, a monkey, a rat, a pig, a bovine and a rabbit, but thepresent invention is not limited thereto.

Also, the HPV-triggered disease may include cervical cancer, anogenitalwarts, verruca, etc.

In addition, the composition according to one exemplary embodiment ofthe present invention may further include a pharmaceutically allowablecarriers. Here, the pharmaceutically allowable carriers includeslactose, glucose, saccharose, sorbitol, mannitol, starch, gum acacia,alginate, gelatine, calcium phosphate, calcium silicate, cellulose,methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone,water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesiumstearate and mineral oil. Also, the composition may further include alubricant, a wetting agent, a flavoring agent, an emulsifying agent anda preservative.

For example, the composition according to one exemplary embodiment ofthe present invention may be directly administered to an individualusing a route of administration such as intravenous, intramuscular,oral, transdermal, intramucosal, intranasal, intratracheal orsubcutaneous administration, but the present invention is not limitedthereto. The composition according to one exemplary embodiment of thepresent invention may be administered indirectly to an individual byadministering the composition into in vitro incubated cells andadministering the incubated cells to the individual. In this case, thecomposition according to one exemplary embodiment of the presentinvention may be administered systemically or locally.

The composition according to one exemplary embodiment of the presentinvention may be formulated into an oral preparation such as a granule,a powder, a solution, a tablet, a capsule or a dry syrup, or aparenteral formulation such as an injectable solution, but the presentinvention is not limited thereto. Preferably, the composition accordingto one exemplary embodiment of the present invention may be prepared inthe form of a solution or an injectable solution.

As the active ingredient, the fusion protein or the recombinantexpression vector according to one exemplary embodiment of the presentinvention may be administered at an effective amount of approximately0.05 to 500 mg/kg, preferably 0.5 to 50 mg/kg. In this case, theadministration may be performed in consideration of a single dose ordivided doses. However, an amount of the active ingredient to beadministered may be determined in consideration of various factors suchas the conditions to be treated, the age and body weight of a patient,and the severity of the conditions, but the present invention is notlimited thereto.

Furthermore, the present invention is directed to providing a method ofpreventing or treating an HPV-triggered disease in an individual in needthereof. Here, the method includes administering an effective amount ofthe composition of the present invention to the individual.

A pharmaceutical composition according to one exemplary embodiment ofthe present invention, use thereof, a method of administering the same,and a dose of the pharmaceutical composition are as described above.

In the method according to one exemplary embodiment of the presentinvention, the individual includes a mammal such as a human, a monkey, arat, a pig, a bovine and a rabbit, but the present invention is notlimited thereto.

Also, the HPV-triggered disease may include cervical cancer, anogenitalwarts, verruca, etc.

Hereinafter, the present invention will be described in further detailwith reference to Examples according to the present invention andComparative Examples which do not fall within the scope of the presentinvention. However, it should be understood that the Examples are notintended to limit the scope of the present invention.

Example 1 Construction of GX-188E DNA

Abbreviations used in the Examples of the present invention are definedas follows. An optimized nucleic acid sequence, “tPa” or “t” refers to asecretory signal sequence of a tissue plasminogen activator, and “F” or“Flt3L” refers to an fms-like tyrosine kinase-3 ligand.

A codon-optimized tPa secretory signal sequence having a nucleic acidsequence set forth in SEQ ID NO: 5 and a codon-optimized Flt3L having anucleic acid sequence set forth in SEQ ID NO: 6 were chemicallysynthesized while the codon-optimized tPa and Flt3L secretory signalsequences were coupled to each other. A terminus of the synthesizedsignal sequence was provided with KpnI (5′) and NheI (3′) restrictionsites to facilitate insertion into a vector. A vector for preparing aDNA vaccine, that is, pGX10 (Korean Patent Publication No. 2003-0047667)was digested with KpnI and NheI restriction enzymes, and then ligatedwith the synthesized tPa-Flt3L signal sequence using ligase to prepare apGX10/tF vector.

A codon-optimized nucleic acid sequence coding for 1^(st) to 85^(th)amino acids of the HPV16 E6, a codon-optimized nucleic acid sequencecoding for 1^(st) to 65^(th) amino acids of the HPV16 E7, acodon-optimized nucleic acid sequence coding for 71^(st) to 158^(th)amino acids of the HPV16 E6, a codon-optimized nucleic acid sequencecoding for 51^(st) to 98^(th) amino acids of the HPV16 E7, acodon-optimized nucleic acid sequence coding for 1^(st) to 85^(th) aminoacids of the HPV18 E6, a codon-optimized nucleic acid sequence codingfor 1^(st) to 65^(th) amino acids of the HPV18 E7, a codon-optimizednucleic acid sequence coding for 71^(st) to 158^(th) amino acids of theHPV18 E6, and a codon-optimized nucleic acid sequence coding for 51^(st)to 105^(th) amino acids of the HPV 18 E7 were chemically synthesizedwhile the codon-optimized nucleic acid sequences were coupled to oneanother (hereinafter referred to as16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C: SEQ ID NO: 4). A terminus ofthe synthesized signal sequence was provided with NheI (5′) and XbaI(3′) restriction sites to facilitate insertion into a vector. ThepGX10/tF vector was digested with NheI and XbaI restriction enzymes, andthen ligated with the synthesized signal sequence,16E6N16E7N16E6C16E7C18E6N18E6N18E7C, using ligase to construct apGX10/tF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C vector. Then, thepGX10/tF16E6N16E7N16E7C18E6N18E7N18E6C18E7C vector was digested withKpnI and XbaI restriction enzymes to separatetF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C, and pGX10 was digested withKpnI and XbaI restriction enzymes, and then ligated withtF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C using ligase to construct apGX27/tF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C vector (hereinafterreferred to as “GX-188E”).

Example 2 Confirmation of Therapeutic Effect of GX-188E on CervicalCancer

To confirm a therapeutic effect of GX-188E on cervical cancer, TC-1tumor cells were subcutaneously injected into C57BL/6 rats at aconcentration of 5×10⁵ cells, and GX-188E was intramuscularly injectedat doses of 50 μg and 100 μg on days 3 and 8, followed by performingelectroporation. A change in volume of tumor cells was observed from theday of injection to day 27, and the spleens of the rats were extractedon Day 36. Then, 1×10⁶ cells were put into a plate coated with 50 μl ofa 5 μg/ml anti-mouse IFN-g antibody (BD Pharmigen, San Diego, Calif.)together with IL-2 and an HPV16 E6 CD8 T cell epitope (E6₄₈₋₅₇;EVYDFAFRDL, Peptron, Korea), an HPV18 E7 CD8 T cell epitope (E7₄₉₋₅₇;RAHYNIVTF, Peptron, Korea), an HPV18 E6 peptide pool, or an HPV18 E7peptide pool, and incubated at 37° C. for 24 hours in a 5% CO₂ incubator(Froma, Minn., USA). The plate was washed with PBST, and a 2 μg/ml IFN-gdetection antibody conjugated with biotin (BD Pharmigen, San Diego,Calif.) was put into the plate at a dose of 50 μl, and incubated at roomtemperature for approximately 3 hours. Subsequently, the plate waswashed with PBST, and streptavidin-alkaline phosphate (AKP) diluted at1:2000 was put into the plate at a dose of 50 μA and incubated at roomtemperature for 1 hour. Then, the plate was washed with PBST, and 66 μlof NBT (Promega, Madison, Wis.) and 33 μl of BCIP (Promega, Madison,Wis.) were added based on 10 ml of an alkaline phosphate buffer.Thereafter, 50 μl of the resulting solution was added and reacted. Theplate was put into a 37° C. incubator and kept for approximately 30minutes to facilitate a color reaction. Then, the plate was washed withdistilled water (D.W.), and colored spots were counted using a reader.

The T cell immune responses specific to HPV16 E6 and E7, and HPV18 E6and E7 were measured in the HPV16 E6 CD8 T cell epitope, the HPV16 E7CD8 T cell epitope, the HPV18 E6 peptide pool and the HPV18 E7 peptidepool using an enzyme-linked immunosorbent spot (ELISPOT) assay. As aresult, it was confirmed that the GX-188E induced a strongantigen-specific immune response, and simultaneously induced an immuneresponse specific to the E6 and E7 of the HPV16 and HPV18 (see FIGS. 1,2, 3 and 4).

Also, it was confirmed that a volume of tumor was significantly reducedin the rats whose TC-1 tumor cells were immunologically treated with theGX-188E, compared with the rats injected with the pGX27 (control) (seeFIG. 5).

INDUSTRIAL APPLICABILITY

The fusion protein according to the present invention can be used as atherapeutic agent for treating HPV-triggered tumors.

The fusion protein according to the present invention, which is preparedto include a fusion polypeptide recombined to transform a 3D structureof E6 and E7 proteins derived from the HPV types 16 and 18, a signalpeptide for secreting the fusion polypeptide outside the cells, and animmunity enhancer peptide present in an individual, can treat theHPV-triggered tumors by inducing a strong immune response specific tothe antigens against the HPV types 16 and 18.

While the invention has been shown and described with reference tocertain exemplary embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the scope of the invention as defined bythe appended claims.

What is claimed is:
 1. A polynucleotide encoding a fusion proteincomprising the sequence of SEQ ID NO:1.
 2. The polynucleotide of claim1, further comprising a nucleic acid sequence encoding an immunityenhancer peptide.
 3. The polynucleotide of claim 2, wherein the immunityenhancer peptide is selected from a CD40 ligand, an fms-like tyrosinekinase-3 (Flt3) ligand, a flagellin, and an OX40.
 4. The polynucleotideof claim 2, wherein the immunity enhancer peptide comprises the sequenceof SEQ ID NO:
 3. 5. The polynucleotide of claim 1, further comprising anucleic acid sequence encoding a signal peptide.
 6. The polynucleotideof claim 5, wherein the signal peptide is selected from a tissueplasminogen activator (“tPa”) signal peptide, a Herpes Simplex Virusglycoprotein Ds (“HSV gDs”) signal peptide, and a growth hormone signalpeptide.
 7. The polynucleotide of claim 5, wherein the signal peptidecomprises the sequence of SEQ ID NO:
 2. 8. The polynucleotide of claim1, which is codon optimized.
 9. The polynucleotide of claim 8,comprising the sequence of SEQ ID NO:
 4. 10. The polynucleotide of claim2, wherein the nucleic acid sequence encoding the immunity enhancerpeptide is codon optimized.
 11. The polynucleotide of claim 5, whereinthe nucleic acid sequence encoding the signal peptide is codonoptimized.
 12. The polynucleotide of claim 11, which comprises thenucleotide sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.13. A vector comprising the polynucleotide of claim
 8. 14. A vectorcomprising the polynucleotide of claim
 9. 15. The vector of claim 14,which is a plasmid.
 16. A host cell comprising the vector of claim 13.17. A composition comprising the polynucleotide of claim 1 and apharmaceutically allowable carrier.
 18. A composition comprising thepolynucleotide of claim 9 and a pharmaceutically allowable carrier. 19.A composition comprising the vector of claim 15 and a pharmaceuticallyallowable carrier.
 20. A fusion protein comprising an amino acidsequence, which is encoded by the polynucleotide of claim
 1. 21. Afusion protein comprising an amino acid sequence, which is encoded bythe polynucleotide of claim
 2. 22. A method of expressing a fusionprotein comprising transfecting the polynucleotide of claim 1 to a hostcell under suitable condition.
 23. A method of producing a fusionprotein, comprising culturing the host cell of claim 16 under suitableconditions.
 24. A method of inducing a T cell immune response to asubject in need thereof comprising administering the polynucleotide ofclaim 1 to the subject, wherein the T cell immune response is against anE6 protein of HPV serotype 16, an E7 protein of HPV serotype 16, an E6protein of HPV serotype 18, and an E7 protein of HPV serotype
 18. 25. Amethod of reducing the volume of a tumor triggered by HPV infection in asubject in need thereof comprising administering the polynucleotide ofclaim 1 to the subject, wherein the tumor volume is reduced after theadministration.